Overexpression of the gene IFIT3 enhances tumor growth, angiogenesis, metastasing and chemoresistance of the pancreas carcinoma cells

Abstract

Background: Pancreas carcinoma belongs to one of the most dangerous tumor disease. Therefore it is very important to understand mechanism leading to change of the low angiogenic and low metastatic tumors to its aggressive counterparts. To address this issue the experimental model consisting of the low metastatic pancreas carcinoma cell line FG and its closely related highly metastatic and proangiogenic cell line L3.6pl has been used. Transcriptome analysis has revealed that expression of gene IFIT3 as well as Interferon α/β receptor has been upregulated in aggressive cell line L3.6pl. Here is the first time described upregulation of this gene in the tumor. Methods: Expression study: real time RT-PCR or western blotting (anti-IFIT3 Ab) using material from FG and L3.6pl cells. Cloning of IFIT3 gene: expression vector pcDNA3.2 V5, Animal experiment: Orthotopic injection of nude mice with FG vector control and with FG cells stable transfected with IFIT3 plasmid. IHC: staining with anti-CD31 antibody. Effect on apoptosis and chemoresistance: Apoptosis assay via propidium iodide staining of both transfected cell lines untreated or treated with different concentration of the antitumor chemotherapeutics. VEGF production of transfectants: ELISA assay of conditioned media. Regulation of IFIT3 transcription: IFIT3-real time RT-PCR upon treatment with NFκB-inhibitor BAY 11–7082. Results: We have shown that highly metastatic and proangiogenic cell line L3.6pl overexpress IFIT3 on the level of RT-PCR, western blotting and IHC as well. Overexpression of IFIT3 in FG cells leads to increased production of VEGF in vitro determined by ELISA. Overexpression of this gene leads to inhibition of apoptosis by untreated cells and to increase of chemoresistance by the cells treated with gemcitabine, 5FU and irrinotecan as well. Tumors initiated by implantation of the FG transfected with IFIT3-plasmid grow significantly faster then tumors obtained by implantation of FG empty vector control cells. Cells transfected with IFIT3 exhibited increased metastatic potential. CD31 staining showed increased microvessels density in tumors derived from FG IFIT3 cells. Treatment with NFκB-inhibitor BAY 11–7082 diminish amount of IFIT3-transcript. Conclusions: Overexpression of IFnα regulated gene IFIT3 leads to increased tumor growth, metastasing, angiogenesis and chemoresistance. This gene is probably regulated via NFκB signalling. Presented results could contribute to understanding of the mechanism of the crosstalk between inflammation and pancreas carcinogenesis.